Fusion polypeptides capable of activating receptors

ABSTRACT

A fusion polypeptide comprising (A)x-M-(A&#39;)y, wherein A and A&#39; are each polypeptides capable of binding a target receptor. The fusion polypeptides of the invention form multimeric proteins which activate the target receptor. A and A&#39; may be each be an antibody or fragment derived from an antibody specific for a target receptor, such as the same or different scFv fragments, and/or a ligand or ligand fragment or derivative capable of binding the target protein, M is a multimerizing component, and X and Y are independently a number between 1-10.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/466,606, filed 15May 2009, which is a continuation-in-part of U.S. Ser. No. 11/035,599,filed 14 Jan. 2005, now U.S. Pat. No. 7,534,604, which claims thebenefit under 35 USC §119(e) of U.S. Provisional 60/536,968 filed 16Jan. 2004, which applications are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to multimeric fusion proteins capable ofactivating a target receptor, methods of producing such fusionpolypeptides, and methods for treating, diagnosing, or monitoringdiseases or conditions in which activation of the target receptor isdesired.

BACKGROUND

The clustering of soluble Eph ligand domains to create multimers capableof activating their cognate receptors is described in U.S. Pat. No.5,747,033. U.S. Pat. No. 6,319,499 recites a method of activating anerythropoietin receptor with an antibody.

BRIEF SUMMARY

The present invention provides multimeric fusion polypeptides capable ofactivating a target receptor requiring multimerization to be activated.The polypeptides of the invention are useful for treating conditions inwhich activation of a target receptor is desirable, as well as having avariety of in vitro and in vivo diagnostic and prognostic uses. Thepolypeptides of the invention may be monospecific or bispecifictetramers exhibiting improved capacity to activate a target receptorrelative to, for example, a target-specific antibody or the naturalligand.

Accordingly, in one aspect the invention provides an isolated nucleicacid molecule which encodes a fusion polypeptide (A)_(x)-M-(A′)_(y),wherein A is a polypeptide specific for a target receptor, M is amultimerizing component, A′ is a polypeptide specific for the sametarget receptor as A, and X and Y are independently a number between1-10.

In a first embodiment, A and A′ are antibodies or antibody fragmentsspecific to the target receptor, and are the same antibody or antibodyfragment specific to a target receptor. In another embodiment, A and A′are different antibodies or antibody fragments specific to the sametarget receptor. Preferably, A and A′ are single chain Fv (ScFv)fragments. When the fusion polypeptide is intended as a humantherapeutic, the invention encompasses humanized antibody or antibodyfragments.

In a second embodiment, A and A′ are ligands or ligand fragmentsspecific for the same target receptor. In a more specific embodiment, Aand A′ are the same or are different ligands or ligand fragmentsspecific to the same target receptor.

In a third embodiment, A is an antibody or antibody fragment specific tothe target receptor, and A′ is a ligand or ligand fragment specific tothe same target receptor. In preferred embodiments, A is an antibody orantibody fragment to a Tie receptor (Tie-1 or Tie-2), and A′ is thefibrinogen domain of a Tie receptor.

In specific embodiments, M is a multimerizing component whichmultimerizes with a multimerizing component on another fusionpolypeptide to form a multimer of the fusion polypeptides. In apreferred embodiment, M is the Fc domain of IgG or the heavy chain ofIgG. The Fc domain of IgG may be selected from the isotypes IgG1, IgG2,IgG3, and IgG4, as well as any allotype within each isotype group.

In another aspect the invention provides a fusion polypeptide comprising(A)_(x)-M-(A′)_(y), wherein A is a polypeptide specific for a targetreceptor, M is a multimerizing component, A′ is a polypeptide specificfor the same target receptor as A, and X and Y are independently anumber between 1-10.

In a first embodiment, A and A′ are antibodies or antibody fragmentsspecific to the target receptor, and are the same antibody or antibodyfragment specific to a target receptor. In another embodiment, A and A′are different antibodies or antibody fragments specific to the sametarget receptor. Preferably, A and A′ are single chain Fv (ScFv)fragments.

In a second embodiment, A and A′ are ligands or ligand fragmentsspecific for the same target receptor. In a more specific embodiment, Aand A′ are different ligands or ligand fragments specific to the sametarget receptor. In another specific embodiment, A and A′ are the sameligand or ligand fragment.

In a third embodiment, A is an antibody or antibody fragment specific tothe target receptor, and A′ is a ligand or ligand fragment specific tothe same target receptor.

In another aspect, the invention provides an activating dimeric fusionpolypeptide comprising two fusion polypeptides of the invention, e.g., adimer formed from two polypeptides of (A)_(x)-M-(A′)_(y) as definedabove. The activating dimers of the invention are capable of binding toand clustering four or more receptors, leading to receptor activation,as compared with the ability of an antibody to cluster no more than tworeceptors.

In one embodiment, the components of the fusion polypeptides of theinvention are connected directly to each other. In other embodiments, aspacer sequence may be included between one or more components, whichmay comprise one or more molecules, such as amino acids. For example, aspacer sequence may include one or more amino acids naturally connectedto a domain-containing component. A spacer sequence may also include asequence used to enhance expression of the fusion polypeptide, providerestriction sites, allow component domains to form optimal tertiary andquaternary structures and/or to enhance the interaction of a componentwith its target receptor. In one embodiment, the fusion polypeptide ofthe invention comprises one or more peptide sequences between one ormore components which is(are) between 1-25 amino acids. Furtherembodiments may include a signal sequence at the beginning oramino-terminus of an fusion polypeptide of the invention. Such a signalsequence may be native to the cell, recombinant, or synthetic.

The components of the fusion polypeptide of the invention may bearranged in a variety of configurations. For example, described from thebeginning or amino-terminus of the fusion polypeptide,(A)_(x)-M-(A′)_(y), (A)_(x)-(A′)_(y)-M, M-(A)_(x)-(A′)_(y),(A′)_(Y)-M-(A)_(X), (A′)_(Y)-(A)_(X)-M, M-(A′)_(Y)-(A)_(X),(A)_(x)-M-(A′)_(y), (A)_(x)-(A′)_(y)-M, M-(A)_(x)-(A′)_(y), etc.,wherein X=1-10 and Y=1-10. In an even more specific embodiment, X=1, andY=1 or X=2 and Y=2.

In one aspect, a Tie receptor (i.e., Tie)-binding protein comprisingfusion polypeptide A-M-A′ is provided, wherein: (a) A comprises aTie-binding fragment of an antibody that binds a Tie-2 receptor (i.e.,Tie-2) on the surface of a cell; (b) A′ is selected from (i) an antibodyfragment that binds Tie-2, and (ii) a fibrinogen domain of Ang-1 (FD1)or Ang-2 (FD2), or Tie-binding fragment thereof; and (c) M is amultimerizing component; wherein binding of A to Tie-2 does not blockbinding of A′ to Tie-2.

In one embodiment, the Tie-binding protein when exposed to a cellbearing a Tie-1, activates Tie-1 and Tie-1 is phosphorylated. In oneembodiment, the Tie-binding protein when exposed to a cell bearing aTie-2 activates the Tie-2 and Tie-2 is phosphorylated. In oneembodiment, the Tie-binding protein when exposed to a cell bearing aTie-1 and a Tie-2, both Tie-1 and Tie-2 are phosphorylated. In oneembodiment, the phosphorylation is more than two-fold higher than aTie-binding protein wherein A and A′ block one another, as measured byimmunoprecipitation of total Tie-1 and/or total Tie-2 and detectingphosphotyrosine. In another embodiment, the phosphorylation is more thanfour-fold. In another embodiment, more than ten-fold.

In one embodiment, the Tie-binding protein binds Tie-2 with higheraffinity than FD1. In one embodiment, exposing a cell bearing a Tie-2 tothe Tie-binding protein results in clustering of more than four Tiereceptors of the cell. In one embodiment, exposing a cell bearing aTie-2 to the Tie-binding protein results in phosphorylation andactivation of a Tie-2 and a Tie-1 of the cell.

In one embodiment, the cell is a mammalian cell selected from a mouse,rat, hamster, monkey, ape, and human cell. In one embodiment, the Tie-2is selected from mouse, rat, hamster, monkey, ape, and human Tie-2. In aspecific embodiment, the Tie-2 is a human Tie-2.

In one embodiment, the Tie-binding protein comprises an A and/or an A′that is an ScFv.

In one embodiment, A (or A′) comprises FD1 or FD2 or a Tie-bindingfragment thereof.

In one embodiment, the FD1 comprises amino acids 515-728 of SEQ IDNO:30. In one embodiment, the FD2 comprises amino acids 515-727 of SEQID NO:31.

In one embodiment, M is selected from the group consisting of an Fcdomain of IgG, and a polypeptide comprising a heavy chain CH2 and CH3constant region. In one embodiment, M is an Fc domain of an IgG thatlacks a CH1 domain. In one embodiment, the Fc domain comprises aminoacids 281-507 of SEQ ID NO:28.

In one embodiment, the Tie-binding protein comprises, from N-terminal toC-terminal (a) a first Tie-binding region comprising a first ScFv thatcomprises a CDR comprising a sequence selected from: amino acids 58-65(HCDR1), 83-90 (HCDR2), 129-139 (HCDR3), 192-201 (LCDR1), 219-221(LCDR2), 258-265 (LCDR3) of SEQ ID NO:28, wherein the first ScFv binds afirst epitope of Tie-2; (b) a multimerizing component; and (c) a secondTie-binding region comprising either (i) a second ScFv that comprises aCDR that binds a second epitope of Tie-2, or (ii) an FD1 or FD2, orTie-binding fragment thereof. In one embodiment, the second ScFvcomprises three HCDRs and three LCDRs.

In one embodiment, the Tie-binding protein comprises, from N-terminal toC-terminal (a) a first Tie-binding region comprising a first ScFv thatcomprises a CDR comprising a sequence selected from: amino acids 58-66(HCDR1), 84-90 (HCDR2), 129-143 (HCDR3), 196-201 (LCDR1), 219-221(LCDR2), 258-265 (LCDR3) of SEQ ID NO:29, wherein the first ScFv binds afirst epitope of Tie-2; (b) a multimerizing component; and (c) a secondTie-binding region comprising either (i) a second ScFv that comprises aCDR that binds a second epitope of Tie-2, or (ii) an FD1 or FD2, orTie-binding fragment thereof. In one embodiment, the second ScFvcomprises three HCDRs and three LCDRs.

In one embodiment, the Tie-binding protein comprises a first polypeptidecomprising first ScFv comprising three HCDRs (amino acids 58-65 (HCDR1),83-90 (HCDR2), 129-139(HCDR3) of SEQ ID NO:28) and three LCDRs (aminoacids 192-201 (LCDR1), 219-221 (LCDR2), 258-265 (LCDR3) of SEQ IDNO:28), wherein the first ScFv binds a first epitope of Tie-2; whereinthe first ScFv is covalently linked (directly or through a linker) tothe N-terminal of an Fc fragment lacking a CH1 domain; and, either (a) asecond ScFv fragment covalently linked to the C-terminal of the Fcfragment lacking the CH1 domain (directly or through a linker), whereinthe second ScFv comprises three HCDRs (amino acids 541-549 (HCDR1),567-573 (HCDR2), 612-626 (HCDR3) of SEQ ID NO:29) and three LCDRs (aminoacids 679-684 (LCDR1), 702-704 (LCDR2), and 741-748 (LCDR3), and whereinthe second ScFv binds a second epitope of Tie-2; or, (b) an FD1 or FD2,or Tie-binding fragment thereof, that does not block binding of thefirst ScFv to Tie-2; and wherein the first polypeptide dimerizes with asecond polypeptide that is identical or substantially identical to thefirst polypeptide such that the first and the second polypeptide form adimer.

In one embodiment, the Tie-binding protein comprises a first polypeptidecomprising (a) a first ScFv comprising three HCDRs (amino acids 58-66(HCDR1), 84-90 (HCDR2), 129-137 (HCDR3) of SEQ ID NO:31) and three LCDRs(amino acids 196-201 (LCDR1), 219-221 (LCDR2), 258-264 (LCDR3) of SEQ IDNO:31), wherein the first ScFv binds a first epitope of Tie-2; andwherein the first ScFv is covalently linked (directly or through alinker) to the N-terminal of an Fc fragment lacking a CH1 domain; and,(b) either (i) a second ScFv fragment covalently linked to theC-terminal of the Fc fragment lacking the CH1 domain (directly orthrough a linker), wherein the second ScFv comprises three HCDRs (aminoacids 58-65 (HCDR1), 83-90 (HCDR2), 129-139 (HCDR3) of SEQ ID NO:28) andthree LCDRs (amino acids 192-201 (LCDR1), 219-221 (LCDR2), and 258-265(LCDR3) of SEQ ID NO:28), and wherein the second ScFv binds the secondepitope of Tie-2; or, (ii) an FD1 or FD2, or Tie-binding fragmentthereof, that does not block binding of the first ScFv to Tie-2; andwherein the first polypeptide dimerizes with a second polypeptide thatis identical or substantially identical to the first polypeptide suchthat the first and the second polypeptide form a dimer.

In one embodiment, the Tie-binding protein comprises, from N-terminal toC-terminal, a polypeptide that comprises (a) a first heavy chainvariable domain (VH) present within the span of amino acids 33-150 ofSEQ ID NO:28; (b) a first light chain variable domain present within thespan of amino acids 166-277 of SEQ ID NO:28; (c) a multimerizingcomponent; (d) a second VH present within the span of amino acids516-637 of SEQ ID NO:28; and, (e) a second VL present within the span ofamino acids 653-762 of SEQ ID NO:28. In one embodiment, themultimerizing component is an immunoglobulin heavy chain constant domain(HC) or an HC that lacks a CH1 domain. In a specific embodiment, themultimerizing component is an IgG heavy chain constant region that lacksa CH1 domain. In a specific embodiment, the multimerizing componentcomprises amino acids 281-507 of SEQ ID NO:28. In one embodiment, theTie-binding protein is a dimer of SEQ ID NO:28.

In one embodiment, framework (FR) regions of the first and/or the secondScFv are human framework regions. In one embodiment, one or more of therecited CDRs are humanized. In a specific embodiment, the FR regions arehuman and one or more of the CDRs are humanized; in another embodimentthe FR regions are human and all CDRs are humanized.

In one embodiment, a Tie-binding protein is provided that comprises ahuman antigen-binding domain that competes with the ScFv of SEQ ID NO:30for binding to Tie-2. In one embodiment, the human antigen-bindingdomain competes with the ScFv contained within amino acids 33-277 of SEQID NO:28 for binding to Tie-2. In one embodiment, a Tie-binding proteinis provided that comprises a human antigen-binding domain that binds theepitope of human Tie-2 which corresponds to the epitope on rTie-2 boundby either ScFv of SEQ ID NO:30 or amino acids 33-277 of SEQ ID NO:28.

In one aspect, a Tie-binding protein comprising fusion polypeptideA-M-A′ is provided, wherein: (a) A comprises a Tie-1-binding fragment ofan antibody that binds a Tie-1 on the surface of a cell; (b) A′ isselected from (i) an antibody fragment that binds a Tie-1, and (ii) afibrinogen domain of Ang-1 (FD1) or Ang-2 (FD2), or Tie-1-bindingfragment thereof; and (c) M is a multimerizing component; whereinbinding of A to Tie-1 does not block binding of A′ to Tie-1.

In one embodiment, the Tie-binding protein when exposed to a cellbearing a Tie-1, activates Tie-1 and Tie-1 is phosphorylated. In oneembodiment, the Tie-binding protein when exposed to a cell bearing aTie-2 activates the Tie-2 and Tie-2 is phosphorylated. In oneembodiment, the phosphorylation is more than two-fold higher than occurswhen the cell is exposed to a Tie-binding protein wherein A and A′ blockone another, as measured by immunoprecipitation of total Tie-1 and/ortotal Tie-2 and detecting phosphotyrosine. In another embodiment, thephosphorylation is more than four-fold. In another embodiment, more thanten-fold.

In one embodiment, the Tie-1-binding protein binds the Tie-1 receptorwith higher affinity than FD1. In one embodiment, exposing a cellbearing a Tie-1 to the Tie-binding protein results in clustering of morethan four Tie-1′s (i.e., more than four Tie-1 receptors) of the cell andalso results in phosphorylation and activation of Tie-1.

In one embodiment, the cell is a mammalian cell selected from a mouse,rat, hamster, monkey, ape, and human cell. In one embodiment, the Tie-1is selected from mouse, rat, hamster, monkey, ape, and human Tie-1. In aspecific embodiment, the Tie-1 is a human Tie-1.

In one embodiment, the Tie-binding protein comprises an A and/or an A′that is an ScFv.

In one embodiment, A (or A′) comprises FD1 or FD2, or a Tie-bindingfragment thereof, that does not block binding of the ScFv to Tie-1. Inone embodiment, the FD1 comprises amino acids 515-728 of SEQ ID NO:30.In one embodiment, the FD2 comprises amino acids 515-727 of SEQ IDNO:31.

In one embodiment, M is selected from the group consisting of an Fcdomain of IgG, and a polypeptide comprising a heavy chain CH2 and CH3constant region. In one embodiment, M is an Fc domain of an IgG thatlacks a CH1 domain. In a specific embodiment, the Fc domain comprisesamino acids 281-507 of SEQ ID NO:28.

In one embodiment, the Tie-binding protein comprises, from N-terminal toC-terminal, (a) a first Tie-1 binding region comprising a first ScFvthat comprises a CDR comprising a sequence selected from: amino acids29-36 (HCDR1), 54-61 (HCDR2), 100-114 (HCDR3), 167-172 (LCDR1), 190-192(LCDR2), 229-237 (LCDR3) of SEQ ID NO:50, wherein the Tie-bindingprotein binds a first epitope of Tie-1; (b) a multimerizing component;and, (c) a second Tie-binding region comprising either (i) a second ScFvthat comprises a CDR that binds a second epitope of Tie-1, or (ii) anFD1 or FD2, or Tie-binding fragment thereof. In one embodiment, thesecond ScFv comprises three HCDRs and three LCDRs.

In one embodiment, the Tie-binding protein comprises, from N-terminal toC-terminal (a) a first Tie-1 binding region comprising a first ScFv thatcomprises a CDR comprising a sequence selected from: amino acids 29-36(HCDR1), 54-61 (HCDR2), 100-111 (HCDR3), 164-169 (LCDR1), 187-189(LCDR2), 226-234 (LCDR3) of SEQ ID NO:51, wherein the Tie-bindingprotein binds a first epitope of Tie-1; (b) a multimerizing component;and, (c) a second Tie-binding region comprising either (i) a second ScFvthat comprises a CDR that binds a second epitope of Tie-1, or (ii) anFD1 or FD2, or Tie-binding fragment thereof. In one embodiment, thesecond ScFv comprises three HCDRs and three LCDRs.

In one embodiment, the Tie-binding protein comprises a first polypeptidecomprising first ScFv comprising three HCDRs (amino acids 29-36 (HCDR1),54-61 (HCDR2), 100-111 (HCDR3) of SEQ ID NO:51) and three LCDRs (aminoacids 164-169 (LCDR1), 187-189 (LCDR2), 226-234 (LCDR3) of SEQ IDNO:51), wherein the first ScFv that binds a first epitope of Tie-1;wherein the first ScFv is covalently linked (directly or through alinker) to the N-terminal of an Fc fragment lacking a CH1 domain; and,either (a) a second ScFv fragment covalently linked to the C-terminal ofthe Fc fragment lacking the CH1 domain (directly or through a linker),wherein the second ScFv comprises three HCDRs (amino acids 509-516(HCDR1), 534-541 (HCDR2), 580-594 (HCDR3) of SEQ ID NO:51) and threeLCDRs (amino acids 647-652 (LCDR1), 670-672 (LCDR2), and 709-717 (LCDR3)of SEQ ID NO:51), wherein the second ScFv binds a second epitope ofTie-1; or (b) an FD1 or FD2, or Tie-binding fragment thereof, that doesnot block binding of the first ScFv to Tie-1; and wherein the firstpolypeptide dimerizes with a second polypeptide that is identical orsubstantially identical to the first polypeptide such that the first andthe second polypeptide form a dimer.

In one embodiment, the Tie-binding protein comprises a first polypeptidecomprising first ScFv comprising three HCDRs (amino acids 29-36 (HCDR1),54-61 (HCDR2), 100-114 (HCDR3) of SEQ ID NO:52) and three LCDRs (aminoacids 167-172 (LCDR1), 190-192 (LCDR2), 229-237 (LCDR3) of SEQ IDNO:52), wherein the first ScFv binds a first epitope of Tie-1; whereinthe first ScFv is covalently linked (directly or through a linker) tothe N-terminal of an Fc fragment lacking a CH1 domain; and, either (a) asecond ScFv fragment covalently linked to the C-terminal of the Fcfragment lacking the CH1 domain (directly or through a linker), whereinthe second ScFv comprises three HCDRs (amino acids 29-36 (HCDR1), 54-61(HCDR2), 100-111 (HCDR3) of SEQ ID NO:51) and three LCDRs (amino acids164-169 (LCDR1), 187-189 (LCDR2), and 226-234 (LCDR3) of SEQ ID NO:51),wherein the second ScFv binds a second epitope of Tie-1; or, (b) an FD1or FD2, or Tie-binding fragment thereof, that does not block binding ofthe first ScFv to Tie-1; and wherein the first polypeptide dimerizeswith a second polypeptide that is identical or substantially identicalto the first polypeptide such that the first and the second polypeptideform a dimer.

In one embodiment, the Tie-binding protein comprises, from N-terminal toC-terminal, a polypeptide that comprises (a) a first heavy chainvariable domain (VH) present within the span of amino acids 4-122 of SEQID NO:51; (b) a first light chain variable domain present within thespan of amino acids 138-244 or 245 of SEQ ID NO:51; (c) a multimerizingcomponent; (d) a second VH present within the span of amino acids484-605 of SEQ ID NO:51; (e) a second VL present within the span ofamino acids 621-728 of SEQ ID NO:51. In one embodiment, themultimerizing component is an immunoglobulin heavy chain constant domain(HC) or an HC that lacks a CH1 domain. In a specific embodiment, themultimerizing component is an IgG that lacks a CH1 domain. In oneembodiment, the multimerizing component comprises amino acids 249-475 ofSEQ ID NO:51). In a specific embodiment, the Tie-binding protein is adimer of SEQ ID NO:51.

In one embodiment, framework (FR) regions of the first and/or the secondScFv are human framework regions. In one embodiment, one or more of therecited CDRs are humanized. In a specific embodiment, the FR regions arehuman and one or more of the CDRs are humanized; in another embodimentthe FR regions are human and all CDRs are humanized.

In one embodiment, a Tie-binding protein is provided that comprises ahuman antigen-binding domain that competes with the ScFv of SEQ ID NO:50for binding to Tie-1. In one embodiment, the human antigen-bindingdomain competes with the ScFv contained within amino acids 4-245 of SEQID NO:51 for binding to Tie-1. In one embodiment, a Tie-binding proteinis provided that comprises a human antigen-binding domain that binds theepitope of human Tie-1 which corresponds to the epitope on rTie-1 boundby either ScFv of SEQ ID NO:50 or amino acids 4-245 of SEQ ID NO:51.

In one embodiment, a human or humanized Tie-binding protein is providedthat comprises, from N-terminal to C-terminal, a human or humanized ScFvthat binds Tie-1, an Fc dimerizing sequence lacking a CH1 domain, and anFD1 (or FD2), wherein the Tie-binding protein when exposed to a cellbearing a human Tie-1 and Tie-2 causes the human Tie-1 and the humanTie-2 to be phosphorylated, wherein the level of phosphorylation of theTie-1 and/or Tie-2 as measured by a phosphotyrosine blot ofimmunoprecipitated total Tie-1 or total Tie-2 is at least two-fold, atleast four-fold, or at least ten-fold higher than that caused byexposing the cell to a monospecific Tie-binding protein or to a mock(e.g., buffer control) treatment. In one embodiment, the human ScFvbinds human Tie-1 within a span of amino acids in the human Tie-1sequence that corresponds, in an optimal alignment of human Tie-1 andrat Tie-1, with a span of amino acids that corresponds with the span ofamino acids of rTie-1 within which the ScFv of SEQ ID NO:52 bindsrTie-1. In one embodiment, the span of amino acids is selected fromfour, six, eight, or ten amino acids. In one embodiment, the epitopebound on human Tie-1 by the human ScFv is the corresponding epitope ofrat Tie-1 with which the ScFv of SEQ ID NO:52 binds.

In one aspect, a Tie-binding protein is provided, comprising apolypeptide that comprises, from N-terminal to C-terminal, (a) a firstScFv that binds an epitope of a Tie receptor; (b) a multimerizingcomponent; and, (c) a second ScFv that binds the same epitope of the Tiereceptor. In one embodiment, the multimerizing component comprises animmunoglobulin heavy chain constant region (HC), in another embodimentthe multimerizing component comprises an HC that lacks a CH1 domain. Inone embodiment, the multimerizing component comprises amino acids280-506 of SEQ ID NO:29.

In one embodiment, the Tie-binding protein binds a Tie-1 receptor. Inone embodiment, the Tie-binding protein binds a Tie-2 receptor.

In one embodiment, the first ScFv and the second ScFv each comprise thesame respective CDRs. In a specific embodiment, the first ScFv and thesecond ScFv are identical.

In one embodiment, the first ScFv comprises three HCDRs (amino acids58-66 (HCDR1), 84-90 (HCDR2), 129-143 (HCDR3) of SEQ ID NO:29) and threeLCDRs (amino acids 196-201 (LCDR1), 219-221 (LCDR2), 258-265 (LCDR3) ofSEQ ID NO:29).

In one embodiment, the first ScFv comprises three HCDRs (amino acids29-36 (HCDR1), 54-61 (HCDR2), 100-114 (HCDR3) of SEQ ID NO:50) and threeLCDRs (amino acids 167-172 (LCDR1), 190-192 (LCDR2), and 229-237 (LCDR3)of SEQ ID NO:50).

In one aspect, a Tie-binding antibody is provided, wherein theTie-binding antibody comprises (a) heavy chain CDR1 (amino acids 58-65),CDR2 (amino acids 83-90) and CDR3 (amino acids 129-139) of SEQ ID NO:28;(b) light chain CDR1 (amino acids 192-201), CDR2 (amino acids 219-221)and CDR3 (amino acids 258-265) of SEQ ID NO:28; and, (c) a light chainconstant domain and a heavy chain constant region.

In one embodiment, the light chain constant domain and the heavy chainconstant domain are human. In one embodiment, the human heavy chainconstant domain is an IgG constant domain, in a specific embodiment, anIgG1 constant domain. In one embodiment, the heavy chain and/or thelight chain CDRs are humanized. In one embodiment, one or more of theframework regions (FRs) are human.

In one embodiment, the Tie-binding antibody comprises the heavy chainvariable region present within the span of amino acids 33-150 of SEQ IDNO:28 and the light chain variable region present within the span ofamino acids 166-277 of SEQ ID NO:28.

In one aspect, a Tie-binding antibody is provided, wherein theTie-binding antibody comprises (a) heavy chain CDR1 (amino acids 58-66),CDR2 (amino acids 84-90) and CDR3 (amino acids 129-143) of SEQ ID NO:29;(b) light chain CDR1 (amino acids 196-201), CDR2 (amino acids 219-221)and CDR3 (amino acids 258-265) of SEQ ID NO:29; and, (c) a light chainconstant domain and a heavy chain constant region.

In one embodiment, the light chain constant domain and the heavy chainconstant domain are human. In one embodiment, the human heavy chainconstant domain is an IgG constant domain, in a specific embodiment, anIgG1 constant domain. In one embodiment, the heavy chain and/or thelight chain CDRs are humanized. In one embodiment, one or more of theframework regions (FRs) are human.

In one embodiment, the Tie-binding antibody comprises the heavy chainvariable region present within the span of amino acids 33-154 of SEQ IDNO:29 and comprises the light chain variable region present within thespan of amino acids 170-276 of SEQ ID NO:29.

In one aspect, a Tie-binding antibody is provided, wherein theTie-binding antibody comprises (a) heavy chain CDR1 (amino acids 29-36),CDR2 (amino acids 54-61) and CDR3 (amino acids 100-114) of SEQ ID NO:50;(b) light chain CDR1 (amino acids 167-172), CDR2 (amino acids 190-192)and CDR3 (amino acids 229-237) of SEQ ID NO:50; and, (c) a light chainconstant domain and a heavy chain constant region.

In one embodiment, the light chain constant domain and the heavy chainconstant domain are human. In one embodiment, the human heavy chainconstant domain is an IgG constant domain, in a specific embodiment, anIgG1 constant domain. In one embodiment, the heavy chain and/or thelight chain CDRs are humanized. In one embodiment, one or more of theframework regions (FRs) are human.

In one embodiment, the Tie-binding antibody comprises the heavy chainvariable region present within the span of amino acids 4-125 of SEQ IDNO:50 and the light chain variable region present within the span ofamino acids 141-248 of SEQ ID NO:50.

In one aspect, a Tie-binding antibody is provided, wherein theTie-binding antibody comprises (a) heavy chain CDR1 (amino acids 29-36),CDR2 (amino acids 54-61) and CDR3 (amino acids 100-111) of SEQ ID NO:51;(b) light chain CDR1 (amino acids 164-169), CDR2 (amino acids 187-189)and CDR3 (amino acids 226-234) of SEQ ID NO:51; and, (c) a light chainconstant domain and a heavy chain constant region sequence.

In one embodiment, the light chain constant domain and the heavy chainconstant region are human. In one embodiment, the human heavy chainconstant region is an IgG constant region, in a specific embodiment, anIgG1 constant domain. In one embodiment, the heavy chain constant regionis an IgG1 constant region that lacks a CH1. In one embodiment, theheavy chain and/or the light chain CDRs are humanized. In oneembodiment, one or more of the framework regions (FRs) are human.

In one embodiment, the Tie-binding antibody comprises the heavy chainvariable region present within the span of amino acids 4-122 of SEQ IDNO:51 and the light chain variable region present within the span ofamino acids 138-244 or 245 of SEQ ID NO:51.

In one aspect, a method for activating a Tie-1 receptor (Tie-1) isprovided, comprising exposing a cell that bears a Tie-1 to aTie-1-binding protein, wherein the Tie-1-binding protein comprises apolypeptide that forms a dimer, wherein the polypeptide comprises, fromN-terminal to C-terminal, (a) a first immunoglobulin heavy chainvariable region (VH1) and light chain variable region (VL1) that binds afirst epitope of Tie-1; (b) a multimerizing component; and, (c) either(i) a second immunoglobulin heavy chain variable region (VH2) and lightchain variable region (VL2) that binds a second epitope of Tie-1, or(ii) an FD1 or FD2 domain; wherein binding of VH1 and VL1 does not blockbinding of VH2 and VL2, and does not block binding of FD1 and FD2.

In one embodiment, the Tie-1 is selected from a mouse, rat, hamster,monkey, ape, and human Tie-1.

In one aspect, a method for activating a Tie-2 receptor (Tie-2) isprovided, comprising exposing a cell that bears a Tie-2 and bears aTie-1 to a Tie-1-binding protein, wherein the Tie-1-binding proteincomprises a polypeptide that forms a dimer, wherein the polypeptidecomprises, from N-terminal to C-terminal, (a) a first immunoglobulinheavy chain variable region (VH1) and light chain variable region (VL1)that binds a first epitope of Tie-1; (b) a multimerizing component; and,(c) either (i) a second immunoglobulin heavy chain variable region (VH2)and light chain variable region (VL2) that binds a second epitope ofTie-1, or (ii) an FD1 or FD2 domain; wherein binding of VH1 and VL1 doesnot block binding of VH2 and VL2, and does not block binding of FD1 andFD2.

In one embodiment, the Tie-1 and Tie-2 are independently selected from amouse, rat, hamster, monkey, ape, and human Tie. In one embodiment, theTie-1 and the Tie-2 are human.

In one aspect, a method for activating a Tie-2 receptor (Tie-2) isprovided, comprising exposing a cell that bears a Tie-2 to aTie-2-binding protein, wherein the Tie-2-binding protein comprises apolypeptide that forms a dimer, wherein the polypeptide comprises, fromN-terminal to C-terminal, (a) a first immunoglobulin heavy chainvariable region (VH1) and light chain variable region (VL1) that binds afirst epitope of Tie-2; (b) a multimerizing component; and, (c) either(i) a second immunoglobulin heavy chain variable region (VH2) and lightchain variable region (VL2) that binds a second epitope of Tie-2, or(ii) an FD1 or FD2 domain; wherein binding of VH1 and VL1 does not blockbinding of VH2 and VL2, and does not block binding of FD1 and FD2.

In one embodiment, the Tie-2 is selected from a mouse, rat, hamster,monkey, ape, and human Tie. In one embodiment, the Tie-2 is human.

In one aspect, a method for activating a Tie-1 receptor (Tie-1) isprovided, comprising exposing a cell that bears a Tie-1 to aTie-2-binding protein, wherein the Tie-2-binding protein comprises apolypeptide that forms a dimer, wherein the polypeptide comprises, fromN-terminal to C-terminal, (a) a first immunoglobulin heavy chainvariable region (VH1) and light chain variable region (VL1) that binds afirst epitope of Tie-2; (b) a multimerizing component; and, (c) either(i) a second immunoglobulin heavy chain variable region (VH2) and lightchain variable region (VL2) that binds a second epitope of Tie-2, or(ii) an FD1 or FD2 domain; wherein binding of VH1 and VL1 does not blockbinding of VH2 and VL2, and does not block binding of FD1 and FD2.

In one embodiment, the Tie-1 and Tie-2 are independently selected from amouse, rat, hamster, monkey, ape, and human Tie. In one embodiment, theTie-1 and Tie-2 are human.

In one aspect, nucleic acids comprising Tie-binding sequences of theTie-binding proteins are provided, as well as nucleic acids encoding thepolypeptides of the Tie-binding proteins.

In another aspect, the invention features a vector comprising a nucleicacid sequence of the invention. The invention further features anexpression vector comprising a nucleic acid of the invention, whereinthe nucleic acid molecule is operably linked to an expression controlsequence. Also provided is a host-vector system for the production ofthe fusion polypeptides of the invention which comprises the expressionvector of the invention which has been introduced into a host cell ororganism, including, but not limited to, transgenic animals, suitablefor expression of the fusion polypeptides.

In another aspect, the invention features a method of producing a fusionpolypeptide of the invention, comprising culturing a host celltransfected with a vector comprising a nucleic acid sequence of theinvention, under conditions suitable for expression of the polypeptidefrom the host cell, and recovering the fusion polypeptide so produced.

In another aspect, the invention features therapeutic methods for thetreatment of a target receptor-related disease or condition, comprisingadministering a therapeutically effective amount of an activating dimerof the invention to a subject in need thereof, wherein the targetreceptor is activated, and the disease or condition is ameliorated orinhibited.

Accordingly, in another aspect, the invention features pharmaceuticalcompositions comprising an activating dimer of the invention with apharmaceutically acceptable carrier. Such pharmaceutical compositionsmay comprise dimeric proteins or nucleic acids which encode them.

In another aspect, a kit is provided that comprises one or moreTie-binding proteins of the invention. In one embodiment, the kitcomprises a Tie-1-binding antibody. In one embodiment, the kit comprisesa Tie-2-binding antibody. In one embodiment, the kit comprises aTie-1-binding antibody and a Tie-2 binding antibody. In one embodiment,the one or more Tie-binding proteins is labelled. In one embodiment, theone or more Tie-binding proteins bind a Tie of an organism selected froma mouse, rat, hamster, monkey, ape, and a human.

Other objects and advantages will become apparent from a review of theensuing detailed description.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

DEFINITIONS

As used herein, the term “target receptor-related condition or disease”generally encompasses a condition of a mammalian host, particularly ahuman host, which is associated with a particular target receptor. Thus,treating a target receptor-related condition will encompass thetreatment of a mammal, in particular, a human, who has symptomsreflective of decreased target receptor activation, or who is expectedto have such decreased levels in response to a disease, condition ortreatment regimen. Treating a target receptor-related condition ordisease encompasses the treatment of a human subject wherein enhancingthe activation of a target receptor with an activating dimer of theinvention results in amelioration of an undesirable symptom resultingfrom the target receptor-related condition or disease. As used herein,an “target receptor-related condition” also includes a condition inwhich it is desirable to alter, either transiently, or long-term,activation of a particular target receptor.

Target Receptors

Examples of target receptors are members of the Eph family (e.g. EphA1,EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3,EphB4, EphB5, EphB6), Tie receptors (e.g. Tie-1 or Tie-2). Suitableligands or fragments thereof include the soluble domain of an ephrin(e.g. ephrin-A1, ephrin-A2, ephrin-A3, ephrin-A4, ephrin-A5, ephrin-B1,ephrin-B2, ephrin-B3), and the fibrinogen domain of an angiopoietin(e.g. angiopoietin-1 (ang-1), ang-2, ang-3, ang-4).

Suitable target receptors are receptors that are activated whenmultimerized. This class of receptors includes, but is not limited to,those that possess an integral kinase domain. Within this class ofintegral kinase receptors are those that form homodimers, or clusters ofthe same receptor, such as Tie-1, Tie-2, EGFR, FGFR, the Trk family andthe Eph family of receptors, and those that form heterodimers, orclusters, such as the VEGF receptors VEGFR1, VEGFR2, the PDGF receptorsPDGFRα and PDGFRβ, and the TGF-β family receptors. Suitable targetreceptors also include, but are not limited to, the class of receptorswith associated kinases. These receptors include those that formhomodimers, or clusters, such as the growth hormone receptor, EPOR andthe G-CSF receptor CD114, and those that form heterodimers, or clusters,such as the GM-CSF receptors GMRα and GMRβ.

Target Receptor-Specific Antibodies and Ligands

In specific embodiments, the activating dimers of the invention compriseone or more immunoglobulin binding domains isolated from antibodiesgenerated against a selected target receptor. The term “immunoglobulin”or “antibody” as used herein refers to a mammalian, including human,polypeptide comprising a framework region from an immunoglobulin gene orfragments thereof that specifically binds and recognizes an antigen,which, in the case of the present invention, is a target receptor orportion thereof. If the intended activating dimer will be used as ahuman therapeutic, immunoglobulin binding regions should be derived fromthe corresponding human immunoglobulins or be a humanizedimmunoglobulin. The human immunoglobulin genes or gene fragments includethe kappa, lambda, alpha, gamma, delta, epsilon, and mu constantregions, as well as the myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.Within each IgG class, there are different isotypes (eg. IgG₁, IgG₂,etc.). Typically, the antigen-binding region of an antibody will be themost critical in determining specificity and affinity of binding.

An exemplary immunoglobulin (antibody) structural unit of human IgG,comprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one light chain (about 25 kD)and one heavy chain (about 50-70 kD). The N-terminus of each chaindefines a variable region of about 100-110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(V_(L)) and “variable heavy chain” (V_(H)) refer to these light andheavy chains respectively.

Antibodies exist as intact immunoglobulins, or as a number ofwell-characterized fragments produced by digestion with variouspeptidases, e.g., F(ab)′₂, Fab′, etc. Thus, the terms immunoglobulin orantibody, as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies, or those synthesizedde novo using recombinant DNA methodologies (e.g., single chain Fv)(ScFv) or those identified using phase display libraries (see, forexample, McCafferty et al. (1990) Nature 348:552-554). In addition, thetarget receptor-binding domain component of the fusion polypeptides ofthe invention include the variable regions of the heavy (V_(H)) or thelight (V_(L)) chains of immunoglobulins, as well as targetreceptor-binding portions thereof. Methods for producing such variableregions are described in Reiter, et al. (1999) J. Mol. Biol.290:685-698.

Methods for preparing antibodies are known to the art. See, for example,Kohler & Milstein (1975) Nature 256:495-497; Harlow & Lane (1988)Antibodies: a Laboratory Manual, Cold Spring Harbor Lab., Cold SpringHarbor, N.Y.). The genes encoding the heavy and light chains of anantibody of interest can be cloned from a cell, e.g., the genes encodinga monoclonal antibody can be cloned from a hybridoma and used to producea recombinant monoclonal antibody. Gene libraries encoding heavy andlight chains of monoclonal antibodies can also be made from hybridoma orplasma cells. Random combinations of the heavy and light chain geneproducts generate a large pool of antibodies with different antigenicspecificity. Techniques for the production of single chain orrecombinant antibodies (U.S. Pat. No. 4,946,778; U.S. Pat. No.4,816,567) can be adapted to produce antibodies used in the fusionpolypeptides, activating dimers and methods of the instant invention.Also, transgenic mice, or other organisms such as other mammals, may beused to express human or humanized antibodies. Alternatively, phagedisplay technology can be used to identify antibodies, antibodyfragments, such as variable domains, and heteromeric Fab fragments thatspecifically bind to selected antigens. Phage display is of particularvalue to isolate weakly binding antibodies or fragments thereof fromunimmunized animals which, when combined with other weak binders inaccordance with the invention described herein, create strongly bindingactivating dimers.

Screening and selection of preferred immunoglobulins (antibodies) can beconducted by a variety of methods known to the art. Initial screeningfor the presence of monoclonal antibodies specific to a target receptormay be conducted through the use of ELISA-based methods or phagedisplay, for example. A secondary screen is preferably conducted toidentify and select a desired monoclonal antibody for use inconstruction of the fusion polypeptides of the invention. Secondaryscreening may be conducted with any suitable method known to the art.

Nucleic Acid Construction and Expression

Individual components of the fusion polypeptides of the invention may beproduced from nucleic acids molecules using molecular biological methodsknown to the art. Nucleic acid molecules are inserted into a vector thatis able to express the fusion polypeptides when introduced into anappropriate host cell. Appropriate host cells include, but are notlimited to, bacterial, yeast, insect, and mammalian cells. Any of themethods known to one skilled in the art for the insertion of DNAfragments into a vector may be used to construct expression vectorsencoding the fusion polypeptides of the invention under control oftranscriptional/translational control signals. These methods may includein vitro recombinant DNA and synthetic techniques and in vivorecombinations (See Sambrook et al. Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory; Current Protocols in MolecularBiology, Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience,NY).

Expression of the nucleic acid molecules of the invention may beregulated by a second nucleic acid sequence so that the molecule isexpressed in a host transformed with the recombinant DNA molecule. Forexample, expression of the nucleic acid molecules of the invention maybe controlled by any promoter/enhancer element known in the art.

Immunoglobulin-Derived Components.

The nucleic acid constructs include regions which encode binding domainsderived from an anti-target receptor antibodies. In general, suchbinding domains will be derived from V_(H) or V_(L) chain variableregions. After identification and selection of antibodies exhibiting thedesired binding characteristics, the variable regions of the heavychains and/or light chains of each antibody is isolated, amplified,cloned and sequenced. Modifications may be made to the V_(H) and V_(L)nucleotide sequences, including additions of nucleotide sequencesencoding amino acids and/or carrying restriction sites, deletions ofnucleotide sequences encoding amino acids, or substitutions ofnucleotide sequences encoding amino acids.

The invention encompasses antibodies or antibody fragments which arehumanized or chimeric. “Humanized” or chimeric forms of non-human (e.g.,murine) antibodies are immunoglobulins, immunoglobulin chains orfragments thereof (such as Fv, Fab, Fab′, F(ab′) 2 or otherantigen-binding subsequences of antibodies) that contain minimalsequences required for antigen binding derived from non-humanimmunoglobulin. They have the same or similar binding specificity andaffinity as a mouse or other nonhuman antibody that provides thestarting material for construction of a chimeric or humanized antibody.Chimeric antibodies are antibodies whose light and heavy chain geneshave been constructed, typically by genetic engineering, fromimmunoglobulin gene segments belonging to different species. Forexample, the variable (V) segments of the genes from a mouse monoclonalantibody may be joined to human constant (C) segments, such as IgG1 andIgG4. Human isotype IgG1 is preferred. A typical chimeric antibody isthus a hybrid protein consisting of the V or antigen-binding domain froma mouse antibody and the C or effector domain from a human antibody.Humanized antibodies have variable region framework residuessubstantially from a human antibody (termed an acceptor antibody) andcomplementarity determining regions (CDR regions) substantially from amouse antibody, (referred to as the donor immunoglobulin). See, Queen etal., Proc. Natl. Acad Sci. USA 86:10029-10033 (1989) and WO 90/07861,U.S. Pat. Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101 and 5,225,539.The constant region(s), if present, are also substantially or entirelyfrom a human immunoglobulin. The human variable domains are usuallychosen from human antibodies whose framework sequences exhibit a highdegree of sequence identity with the murine variable region domains fromwhich the CDRs were derived. The heavy and light chain variable regionframework residues can be derived from the same or different humanantibody sequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies or can be consensus sequences ofseveral human antibodies. See WO 92/22653. Certain amino acids from thehuman variable region framework residues are selected for substitutionbased on their possible influence on CDR conformation and/or binding toantigen. Investigation of such possible influences is by modeling,examination of the characteristics of the amino acids at particularlocations, or empirical observation of the effects of substitution ormutagenesis of particular amino acids. For example, when an amino aciddiffers between a murine variable region framework residue and aselected human variable region framework residue, the human frameworkamino acid should usually be substituted by the equivalent frameworkamino acid from the mouse antibody when it is reasonably expected thatthe amino acid: (1) noncovalently binds antigen directly; (2) isadjacent to a CDR region; (3) otherwise interacts with a CDR region(e.g. is within about 6 A of a CDR region), or (4) participates in theV_(L)-V_(H) interface. Other candidates for substitution are acceptorhuman framework amino acids that are unusual for a human immunoglobulinat that position. These amino acids can be substituted with amino acidsfrom the equivalent position of the mouse donor antibody or from theequivalent positions of more typical human immunoglobulins. Othercandidates for substitution are acceptor human framework amino acidsthat are unusual for a human immunoglobulin at that position. Thevariable region frameworks of humanized immunoglobulins usually show atleast 85% sequence identity to a human variable region frameworksequence or consensus of such sequences.

Fully human antibodies may be made by any method known to the art. Forexample, U.S. Pat. No. 6,596,541 describes a method of generating fullyhuman antibodies. Briefly, initially a transgenic animal such as a mouseis generated that produces hybrid antibodies containing human variableregions (VDJ/VJ) and mouse constant regions. This is accomplished by adirect, in situ replacement of the mouse variable region (VDJ/VJ) geneswith their human counterparts. The mouse is then exposed to humanantigen, or an immunogenic fragment thereof. The resultant hybridimmunoglobulin loci will undergo the natural process of rearrangementsduring B-cell development to produce hybrid antibodies having thedesired specificity. The antibody of the invention is selected asdescribed above. Subsequently, fully-human antibodies are made byreplacing the mouse constant regions with the desired humancounterparts. Fully human antibodies can also be isolated from mice orother transgenic animals such as cows that express human transgenes orminichromosomes for the heavy and light chain loci. (Green (1999) JImmunol Methods. 231:11-23 and Ishida et al (2002) Cloning Stem Cells.4:91-102). Fully human antibodies can also be isolated from humans towhom the protein has been administered. Fully human antibodies can alsobe isolated from immune compromised mice whose immune systems have beenregenerated by engraftment with human stem cells, splenocytes, orperipheral blood cells (Chamat et al (1999) J Infect Dis. 180:268-77).To enhance the immune response to the protein of interest one canknockout the gene encoding the protein of interest in thehuman-antibody-transgenic animal.

Receptor-Binding Domains.

In accordance with the invention, the nucleic acid constructs includecomponents which encode binding domains derived from target receptorligands. After identification of a ligand's target receptor-bindingdomain exhibiting desired binding characteristics, the nucleic acid thatencodes such domain is used in the nucleic acid constructs. Such nucleicacids may be modified, including additions of nucleotide sequencesencoding amino acids and/or carrying restriction sites, deletions ofnucleotide sequences encoding amino acids, or substitutions ofnucleotide sequences encoding amino acids.

The nucleic acid constructs of the invention are inserted into anexpression vector or viral vector by methods known to the art, whereinthe nucleic acid molecule is operatively linked to an expression controlsequence. Also provided is a host-vector system for the production ofthe fusion polypeptides and activating dimers of the invention, whichcomprises the expression vector of the invention, which has beenintroduced into a suitable host cell. The suitable host cell may be abacterial cell such as E. coli, a yeast cell, such as Pichia pastoris,an insect cell, such as Spodoptera frugiperda, or a mammalian cell, suchas a COS, CHO, 293, BHK or NS0 cell.

The invention further encompasses methods for producing the activatingdimers of the invention by growing cells transformed with an expressionvector under conditions permitting production of the fusion polypeptidesand recovery of the activating dimers formed from the fusionpolypeptides. Cells may also be transduced with a recombinant viruscomprising the nucleic acid construct of the invention.

The activating dimers may be purified by any technique which allows forthe subsequent formation of a stable dimer. For example, and not by wayof limitation, the activating dimers may be recovered from cells eitheras soluble polypeptides or as inclusion bodies, from which they may beextracted quantitatively by 8M guanidinium hydrochloride and dialysis.In order to further purify the activating dimers, conventional ionexchange chromatography, hydrophobic interaction chromatography, reversephase chromatography or gel filtration may be used. The activatingdimers may also be recovered from conditioned media following secretionfrom eukaryotic or prokaryotic cells.

Screening and Detection Methods

The activating dimers of the invention may also be used in in vitro orin vivo screening methods where it is desirable to detect and/or measuretarget receptor levels. Screening methods are well known to the art andinclude cell-free, cell-based, and animal assays. In vitro assays can beeither solid state or soluble. Target receptor detection may be achievedin a number of ways known to the art, including the use of a label ordetectable group capable of identifying an activating dimer which isbound to a target receptor. Detectable labels are well developed in thefield of immunoassays and may generally be used in conjunction withassays using the activating dimer of the invention.

Therapeutic Methods

The ability of the activating dimers of the invention to exhibit highaffinity binding for their receptors makes them therapeutically usefulfor efficiently activating their receptors. Thus, it certain instancesit may be to increase the effect of endogenous ligands for targetreceptors, such as, for example, the ephrins. For example, in the areaof nervous system trauma, certain conditions may benefit from anincrease in ephrin responsiveness. It may therefore be beneficial toincrease the binding affinity of an ephrin in patients suffering fromsuch conditions through the use of the compositions described herein.

The invention herein further provides for the development of anactivating dimer described herein as a therapeutic for the treatment ofpatients suffering from disorders involving cells, tissues or organswhich express the Tie-2 receptor. Such molecules may be used in a methodof treatment of the human or animal body, or in a method of diagnosis.

The target receptor known as Tie-2 receptor has been identified inassociation with endothelial cells and, as was previously demonstrated,blocking of agonists of the receptor such as Tie-2 ligand 1 (Ang1) hasbeen shown to prevent vascularization. Accordingly, activating dimers ofthe invention wherein the target receptor is Tie-2 may be useful for theinduction of vascularization in diseases or disorders where suchvascularization is indicated. Such diseases or disorders would includewound healing, ischemia and diabetes. The ligands may be tested inanimal models and used therapeutically as described for other agents,such as vascular endothelial growth factor (VEGF), another endothelialcell-specific factor that is angiogenic. Ferrara et al. U.S. Pat. No.5,332,671 issued Jul. 26, 1994. Ferrara et al. describe in vitro and invivo studies that may be used to demonstrate the effect of an angiogenicfactor in enhancing blood flow to ischemic myocardium, enhancing woundhealing, and in other therapeutic settings wherein neoangiogenesis isdesired. According to the invention, such a Tie-2 specific activatingdimer may be used alone or in combination with one or more additionalpharmaceutically active compounds such as, for example, VEGF or basicfibroblast growth factor (bFGF).

Methods of Administration

Methods known in the art for the therapeutic delivery of agents such asproteins or nucleic acids can be used for the therapeutic delivery of anactivating dimer or a nucleic acid encoding an activating dimer of theinvention for activating target receptors in a subject, e.g., cellulartransfection, gene therapy, direct administration with a deliveryvehicle or pharmaceutically acceptable carrier, indirect delivery byproviding recombinant cells comprising a nucleic acid encoding anactivating dimer of the invention.

Various delivery systems are known and can be used to administer theactivating dimer of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987,J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part ofa retroviral or other vector, etc. Methods of introduction can beenteral or parenteral and include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, pulmonary,intranasal, intraocular, epidural, and oral routes. The compounds may beadministered by any convenient route, for example by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local. Pulmonary administration canalso be employed, e.g., by use of an inhaler or nebulizer, andformulation with an aerosolizing agent.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositionscomprising an activating dimer of the invention and a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Suitablepharmaceutical excipients include starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. (See, forexample, “Remington's Pharmaceutical Sciences” by E. W. Martin.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Where necessary, thecomposition may also include a solubilizing agent and a local anestheticsuch as lidocaine to ease pain at the site of the injection. Where thecomposition is to be administered by infusion, it can be dispensed withan infusion bottle containing sterile pharmaceutical grade water orsaline. Where the composition is administered by injection, an ampouleof sterile water for injection or saline can be provided so that theingredients may be mixed prior to administration.

The active agents of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

Kits

The invention also provides a pack or kit (e.g., a pharmaceutical packor kit) comprising one or more containers filled with at least oneactivating dimer of the invention. The kits of the invention may be usedin any applicable method, including, for example, diagnostically.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflects(a) approval by the agency of manufacture, use or sale for humanadministration, (b) directions for use, or both.

Transgenic Animals

The invention includes transgenic non-human animals expressing a fusionpolypeptide of the invention. A transgenic animal can be produced byintroducing nucleic acid into the male pronuclei of a fertilized oocyte,e.g., by microinjection, retroviral infection, and allowing the oocyteto develop in a pseudopregnant female foster animal. Any of theregulatory or other sequences useful in expression vectors can form partof the transgenic sequence. A tissue-specific regulatory sequence(s) canbe operably linked to the transgene to direct expression of thetransgene to particular cells. A transgenic non-human animal expressingan fusion polypeptide of the invention is useful in a variety ofapplications, including as a means of producing such fusion proteinsFurther, the transgene may be placed under the control of an induciblepromoter such that expression of the fusion polypeptide may becontrolled by, for example, administration of a small molecule.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Production of Anti-Tie-2 Hybridomas

Five 8-weeks old Balb/c mice were first immunized with purified humanTie-2-Fc (hTie-2-Fc); each mouse was injected subcutaneously with 200 μlemulsion containing 100 μg purified hTie-2-Fc protein and 100 μlFreund's complete adjuvant. Fifteen days after the primary injection,each mouse received subcutaneous injection of 200 μl emulsion containing100 μg purified hTie-2-Fc in 100 μl PBS and 100 μl Freund's incompleteadjuvant. This injection was repeated for the five mice seven dayslater. One mouse was used for generation of hybridomas against hTie-2.Each of the four remaining mice were given subcutaneous injections of200 μl emulsion each containing 100 μg purified rat Tie-2-Fc (rTie-2-Fc)in 100 μl PBS and 100 μl Freund's incomplete adjuvant six months afterthe primary injection of hTie-2-Fc. Eleven days later, the immuneresponse of the mice to rTie-2-Fc was boosted by subcutaneous injectionof 200 μl of emulsion containing 100 μg purified rTie-2-Fc in 100 μl PBSand 100 μl Freund's incomplete adjuvant for each mouse. Mouse sera werecollected from tail veins three days after the injection, then theantibody titers against rTie-2-Fc were determined by ELISA. The two micewith the highest titers were given a final boost by tail vein injectionof 100 μg purified rTie-2-Fc in 100 μl PBS. The mice were sacrificedthree days later and their spleen cells were collected for fusion withSp2/0-Ag14 cells.

To generate hybridomas, mouse spleen cells were fused with Sp2/0-Ag14myeloma cells using polyethylene glycol (PEG). Briefly, after thespleens were aseptically removed from the mice, one tip of each spleenwas cut open and spleen cells collected. The spleen cells were washedtwice with D-MEM and cell numbers were counted using a hemocytometer.2×10⁸ spleen cells were combined with 3×10⁷ Sp2/0-Ag14 cells that werein log growth stage. The cell mix was washed with 30 mls D-MEM. 1 ml 50%PEG at 37° C. was slowly added to the cell pellet while stirring. D-MEMwas added to the mix to bring the volume to 10 mls. The cells were spundown at 400×g for 10 minutes. After removal of supernatant, the cellswere gently resuspended in 20 mls growth medium containing 60% D-MEMwith 4.5 g/L glucose, 20% FCS, 10% NCTC109 medium, 10% hybridoma cloningfactor, 1 mM oxaloacetate, 2 mM glutamine, 0.2 units/ml insulin, and 3μM glycine. The cells were transferred to two T225 flasks, eachcontaining 100 mls of the growth medium and were put into a tissueculture incubator. On the next day, 1× HAT was added to the medium toselect against the myeloma cells that were not fused. Nine days afterthe fusion, the cultures were replenished with fresh medium. Human IgGwas added to the cultures at 1 mg/ml. On the tenth day after the fusion,2.6×10⁷ fused cells were stained sequentially with 1 μg/mlbiotin-rTie-2-Fc for one hour and 2.5 μg/ml phycoerythrin(PE)-conjugated streptavidin for 45 minutes in growth medium at roomtemperature. As a control, 1×10⁶ fused cells were stained with 2.5 μg/mlPE-streptavidin for 45 minutes at room temperature. The cells werewashed with 10 ml PBS after each stain. After staining, the cells wereresuspended in PBS with 0.1% FCS and were analyzed by flow cytometry ona MoFlo (Cytomation). A population of cells (4% total cells) stainedwith both biotin rTie-2-Fc and PE-streptavidin exhibited fluorescencehigher than the unstained cells and the cells stained withPE-streptavidin alone. The cells in this 4% gate were cloned by sortingsingle cells into two 96-well plates containing 200 μl growth medium perwell. The cells were cultured for 10 days before splitting into two setsof 96-well plates. Cells in one set of plate were processed for RT-PCRof mouse IgG heavy chain variable region following by sequencing. Theclones were grouped into 14 bins, with members of each bin havingidentical sequence in their heavy chain variable region. Conditionedmedium of hybridoma cells in each bin was tested for its ability tostimulate phosphorylation of rTie-2 in cultured rat aortic endothelialcells (RAECs).

Antibodies from two hybridomas, B2 and A12A, were chosen for furtherstudy because they were active in phosphorylation of Tie-2 in RAECs, anddid not compete for binding to rTie-2 as determined by BIAcore analysis.In addition, these antibodies did not block binding of derivatives ofangiopoietin-1 (Ang1) and angiopoietin-2 (Ang2), the natural ligands ofTie-2.

Example 2 Construction of ScFvs (B2 and A12A)

Generally, antibody variable regions from hybridomas expressingantibodies specific for rTie-2 were cloned by first determining the DNAsequence of RT-PCR products using primers specific for mouse antibodyvariable regions, then using specific primers based on the determinedsequence in order to amplify DNA fragments encoding ScFvs. The ScFv DNAfragments were cloned such that they could be cassette exchanged withmultiple plasmids to yield all combinations of activating dimers. Forexample, one amplified ScFv fragment could be fused to a signal sequenceat the N-terminus and to a coding sequence for the IgG Fc domain at theC-terminus, or it could be fused to the C-terminus of an IgG Fc codingsequence such that the 3′ end of the ScFv coding sequence contained atranslation stop codon.

The B2 hybridoma was found to express an antibody capable of inducingphosphorylation of the Tie-2 receptor in RAECs. Total RNA was isolatedfrom this hybridoma using the promega SV96 Total RNA Isolation System(Promega) and variable heavy cDNA was synthesized using the QiagenOne-Step RT-PCR system (Qiagen) with heavy chain primers from the Ratnerprimer set (Wang et al. (2000) J. Immunol. Methods 233:167) thatincluded equimolar amounts of the 5′ primers (SEQ ID NO:1-7) and the 3′primer (SEQ ID NO:8). Similarly, the light chain variable regions wereamplified from cDNA using equimolar amounts of the light chain-specificprimers (SEQ ID NO:9 and 10). The amplified variable region fragmentswere cloned into the pCR2.1-TOPO vector (Invitrogen) and the DNAsequences were determined. Based on the determined variable regionsequences for the B2 antibody, the variable heavy sequence was PCRamplified using the pCR2.1-TOPO cloned variable region as template andan equimolar mix of 5′ and 3′ primers (SEQ ID NO: 19 and SEQ ID NO: 20).The variable light sequence was PCR amplified using a similar strategy.The pCR2.1-TOPO cloned variable region was used as template and anequimolar mix of 5′ and 3′ primers (SEQ ID NO:21 and SEQ ID NO:22). Thevariable regions were joined by a (G4S)3 linker; ScFv genes wereassembled and PCR amplified using an equimolar mix of the above specificvariable heavy and variable light PCR products and an equimolar mix of5′ B2 heavy primer (SEQ ID NO:19) and the 3′ light primer (SEQ IDNO:22). PCR product was cloned into Invitrogen pCR2.1-TOPO (Invitrogen)to yield pRG1039. The sequence was confirmed before sub-cloning the 744bp AscI/SrfI to fuse the ScFv gene to the N-terminus of a DNA encodingthe human IgG1 Fc fragment (hFc), or the 753 bp AscI/NotI restrictionfragments to fuse the same ScFv to the C-terminus of a DNA encoding hFc.

The A12A hybridoma was also found to express an antibody capable ofinducing phosphorylation of the Tie-2 receptor in RAECs. Total RNA wasisolated from this hybridoma using the Quick Prep mRNA purification kit(Amersham Pharmacia Biotech) and variable heavy cDNA was synthesizedusing the Qiagen One-Step RT-PCR system, with equimolar amounts ofprimers from the from the Wright primer set (Morrison et al. (1995)Antibody Engineering, second edition, Borrebaeck, C. K. A. editor267-293) that included the 5′ heavy chain primers (SEQ ID NO:11-13) andthe 3′ primer (SEQ ID NO:8). Similarly, the light chain variable regionswere amplified from cDNA with equimolar amounts of the 5′ heavy chainprimers (SEQ ID NO:14-18) and the 3′ primer (SEQ ID NO:10). Theamplified variable region fragments were cloned into the pCR2.1-TOPOvector (Invitrogen) and the DNA sequences were determined.

Based on the determined variable region sequences for the A12A antibody,the variable heavy sequence was PCR amplified using the pCR2.1-TOPOcloned variable region as template and an equimolar mix of 5′ and 3′primers (SEQ ID NO:23 and SEQ ID NO:24). The variable light sequence wasPCR amplified using a similar strategy. The pCR2.1-TOPO cloned variableregion was used as template and an equimolar mix of 5′ and 3′ primers(SEQ ID NO:25 and SEQ ID NO:26). The variable regions were joined by a(G₄S)₃ linker; ScFv genes were assembled and PCR amplified using anequimolar mix of the above specific variable heavy and variable lightPCR products and an equimolar mix of 5′ A12A heavy primer (SEQ ID NO:23)and the 3′ light primer (SEQ ID NO:26). PCR product was cloned intoInvitrogen pCR2.1-TOPO to yield pRG1090. The sequence was confirmedbefore sub-cloning the 747 bp AscI/SrfI to fuse the ScFv gene to theN-terminus of a DNA encoding the hFc fragment.

Example 3 Construction of Monospecific and Bispecific Activating Dimers

The general scheme for constructing both monospecific and bispecifictetravalent activating dimers was based on the ability of either the B2or A12A ScFv genes to be inserted between the murine ROR1 signalsequence (SEQ ID NO:27) and the gene encoding hFc (nucleotides 85 to 765of GenBank accession #X70421) when cut with one set of restrictionenzymes, or after the hFc gene if cut with a different set of enzymes.This design of the ScFv genes allowed the exchange of ScFv cassettesamong plasmids to obtain different combinations of ScFv and hFc usingstandard known methods. All constructs have an optional three amino acidlinker (spacer) between the cleavage site of the signal peptide and thestart of the ScFv gene, resulting from engineering a restriction siteonto the 5′ end of the ScFv genes. Similarly, fusion to the aminoterminus of the hFc gene was facilitated by a three amino acid sequence(Gly-Pro-Gly), and fusion to the carboxy terminus of the hFc gene wasfacilitated by an eight amino acid sequence consisting of the residuesGly₄-Ser-Gly-Ala-Pro (SEQ ID NO:32) As a consequence of the terminalrestriction site linkers on the ScFv genes, all constructs that have acarboxy terminal ScFv end with the amino acids Gly-Pro-Gly.

Two types of svFc-based chimeric molecules were constructed to assessthe ability of ScFv-based molecules to activate the rTie-2 receptor. Onetype of molecule used a single ScFv fused to both the N-terminus and theC-terminus of hFc, the consequence of which was a monospecifictetravalent molecule capable of binding rTie-2. This molecule wasexpected to be capable of simultaneously binding four rTie-2 molecules.The plasmid pTE586 encodes the gene for ScFv_(B2)-Fc-ScFv_(B2) (SEQ IDNO: 29) whose secretion is directed by the mROR1 signal peptide. Theexpression of ScFv_(B2)-Fc-ScFv_(B2) in pTE586 was directed by theCMV-MIE promoter when transfected into CHO cells. This protein waseasily purified by Protein A-Sepharose affinity chromatography.

Construction of an ScFv-Fc-ScFv molecule wherein the two ScFv domainsare derived from two different non-competing anti-rTie-2 antibodieswould yield a molecule capable of clustering more than four receptors,in contrast to the ScFv_(B2)-Fc-ScFv_(B2) described above, which cancluster only four receptors. It was determined by BIAcore analysis thatthe binding of the B2 antibody did not block binding of A12A to rTie-2,and A12A binding first did not block binding of B2. Consequently, ScFvmolecules made from these antibodies should be capable of clusteringmore than four receptors. To construct a bispecific tetravalentScFv-based molecule, the ScFv_(A12A) gene was used in combination withthe ScFv_(B2) gene to yield ScFv_(A12A)-Fc-ScFv_(B2) (SEQ ID NO: 28).The plasmid pTE585 encodes the gene for ScFv_(A12A)-Fc-ScFV_(B2) and hasthe mROR1 signal peptide and CMV-MIE promoter when transfected into CHOcells. Both ScFv_(B2)-Fc-ScFv_(B2) and ScFv_(A12A)-Fc-ScFv_(B2) wereexpressed in CHO cells, and purified by Protein A-Sepharose affinitychromatography.

Example 4 Assays

Antibodies to rTie-2, and chimeric molecules related to theseantibodies, were evaluated for their ability to induce phosphorylationof Tie-2 in cultured rat aortic endothelial cells. Confluent RAECs,between passage 3 and 6 (Vec Technologies), were grown in MCDB-131 media(Vec Technologies) on 0.2% gelatin coated T-75 flasks. Cells werestarved for 2 hrs. in serum-free DME-Hi glucose medium (IrvineScientific) prior to incubation at 37° C. for 5 min. in 1.5 mlserum-free DME-Hi glucose medium with 0.1% BSA and the challengemolecule. The challenge medium was then removed and cells were lysed in20 mM Tris, pH 7.6, 150 mMNaCl, 50 mM NaF, 1 mM Na orthovanadate, 5 mMbenzamidine, 1mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS,with 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mM PMSF. Tie-2 wasimmunoprecipitated by incubating the lysates at 4° C. for 16 hrs. with 5μg anti-Tie-2 mouse monoclonal antibody KP-m33, 10 μg biotinylatedanti-mouse IgG (Jackson Laboratories), and 100 μl of neutravidin beads(Pierce). Beads were collected by centrifugation, washed 3 times withRIPA buffer, and bound proteins were eluted with 40 μl of 5× Laemmlibuffer with 10% B-mercaptoethanol by heating at 100° C. for 5 min. AfterSDS-gel electrophoresis on a 4-12% Tris/glycine polyacrylamide gel(Novex), proteins were transferred to PVDF membranes and probed withmouse anti-phophotyrosine monoclonal antibody 4G10 (Upstate) thendetected using goat anti-mouse IgG-HRP conjugate (Pierce) followed byECL reagent (Amersham). The ability to induce Tie-2 phosphorylation inRAECs was determined for each activating dimer. Activity was evaluatedby comparison to the level of stimulation obtained with FD1-Fc-FD1(BA1)—a chimeric protein shown to be as active as Ang1 in binding andactivation Tie-2 (Davis et al. (2003) Nature Struct. Biol. 10:38-44)(FD1 or FD2=human fibrinogen domain of Ang1 or Ang2, respectively).Maximum stimulation (ECmax) of Tie-2 in RAECs was observed when BA1 wasused at about 0.5 to 1.0 μg/ml, and phosphorylation levels in mocktreated cells were low. Similarly, the ECmax of ScFv_(B2)-Fc-ScFv_(B2),ScFv_(A12A)-Fc-ScFv_(B2), ScFv_(B2)-Fc-FD1, and ScFv_(B2)-Fc-FD2 wereabout 0.5 to 1.0 μg/ml. In all cases, the ScFv-based molecules werecapable of inducing a higher phosphorylation signal than observed forthe related native antibodies isolated from hybridoma conditioned media.

Purified ScFv_(B2)-Fc-ScFv_(B2) and ScFv_(A12A)-Fc-ScFv_(B2) werecharacterized for their ability to bind rTie-2 and inducephosphorylation. Binding to rTie-2 was determined by BIAcore analysis.Both the monospecific and the bispecific activating dimers were found tohave significantly higher affinity for rTie-2 than FD1-Fc-FD1. Inaddition, both ScFv_(B2)-Fc-ScFv_(B2) and ScFv_(A12A)-Fc-ScFv_(B2) wereable to stimulate phosphorylation of rTie-2 in RAECs comparable toFD1-Fc-FD1.

Example 5 Construction of ScFv/Ligand Activating Dimers

Bispecific tetravalent molecules were constructed to include both Tie-2specific ScFv and FD1 or FD2. The chimeric molecules were made by fusingthe gene encoding ScFv_(B2) to the N-terminus of hFc and the geneencoding Ang1 FD (Phe283 to Phe498 of GenBank accession #Q15389) or Ang2FD (Phe281 to Phe496 of GenBank accession #O15123) to the C-terminus.Plasmid pTE514 encodes the gene for ScFv_(B2)-Fc-FD1 (SEQ ID NO: 30) andcontained the mROR1 signal peptide and CMV-MIE promoter. Plasmid pTE614encodes the gene for ScFv_(B2)-Fc-FD2 (SEQ ID NO: 31) and contained themROR1 signal peptide and CMV-MIE promoter. Similar toScFv_(B2)-Fc-ScFv_(B2) and ScFv_(A12A)-Fc-ScFv_(B2) the proteinsexpressed from pTE514 and pTE614 had a Gly-Ala-Pro linker between themROR1 signal peptide and the ScFv_(B2), a Gly-Pro-Gly linker between theN-terminal ScFv_(B2) and hFc and a Gly₄-Ser-Gly-Ala-Pro linker (SEQ IDNO:32) between the C-terminus of hFc and the N-terminus of the Ang FDs.Both ScFv_(B2)-Fc-FD1 and ScFv_(B2)-Fc-FD2 were expressed and purifiedas described above.

Purified ScFv_(B2)-Fc-FD1 and ScFv_(B2)-Fc-FD2 were characterized fortheir ability to bind rTie-2 and induce phosphorylation as described inabove. As determined by BIAcore analysis, the chimeric activating dimerScFv_(B2)-Fc-FD1 was found to have significantly higher affinity forrTie-2 (0.04 nM) than FD1-Fc-FD1 (2 nM). Moreover, both ScFv_(B2)-Fc-FD1and ScFv_(B2)-Fc-FD2 were able to stimulate phosphorylation of rTie-2 inRAECs comparable to FD1-Fc-FD1.

Example 6 Construction of Fully Human Activating Dimers

Bispecific tetravalent molecules are formed from dimerized fusionconstructs of the invention which include either two ScFvs derived fromhuman antibodies specific for hTie-2 or one ScFv derived from a humanantibody specific for hTie-2 and human FD1 or FD2. Human ScFvs specificfor hTie-2 are obtained by methods known to the art and as describedabove. In one embodiment, human ScFvs are obtained recombinantly asdescribed in Reiter et al. (1999) J. Mol. Biol. 290:685-698 andGilliland et al. (1996) Tissue Antigens 47(1):1-20.

Example 7 Construction of ScFvs (1-1F11 and 2-1G3)

Anti-rTie-1 hybridomas were produced following the procedures describedabove for the production of anti-rTie-2 hybridomas. Briefly, mice wereimmunized three times with purified rat Tie-1-Fc protein and Freund'sadjuvant. Spleen cells from the mouse with the highest anti-Tie-1antibody titer were fused with Sp2/0-Ag14 myeloma cells usingpolyethylene glycol (PEG). After fusion, the cells were cultured in twoT225 flasks. HAT was added to the cultures on the next day. Nine daysafter the fusion, the cultures were replenished with fresh medium. HumanIgG was added to the cultures at 1 mg/ml. On the tenth day after thefusion, the HAT-resistant cells were stained sequentially with 1 μg/mlbiotin-rat Tie-1-Fc for one hour and 2.5 μg/ml phycoerythrin(PE)-conjugated streptavidin for 45 minutes in growth medium at roomtemperature. After staining, the cells were analyzed by flow cytometry.Cells that bound rTie-1-Fc were cloned by sorting single cells into96-well plates. The 96-well plate cultures were split into two sets tendays after sorting. RT-PCR of mouse IgG heavy chain variable regionfollowed by sequencing were performed on one set of the 96-well platecultures. Clones with unique IgG heavy chain variable region sequenceswere identified and expanded for the production of anti-rTie-1antibodies. Antibodies were tested for binding rTie-1 protein and twoclones, 1-1F11 and 1-2G3, were chosen for more detailed study.

The 1-1F11 hybridoma was found to express an antibody capable ofinducing phosphorylation of the Tie-1 receptor in RAECs. Messenger RNAwas isolated and variable heavy cDNA synthesized as described above withheavy chain primers from the Wright primer set (Morrison et al. (1995)Antibody Engineering, second edition, Borrebaeck, C. K. A. editor267-293) that included the 5′ heavy chain primers (SEQ ID NO:35-37) andthe 3′ primer (SEQ ID NO:33). Similarly, the light chain variableregions were amplified from cDNA with equimolar amounts of the 5′ lightchain primers (SEQ ID NO:38-41) and the 3′ primer (SEQ ID NO:34). Theamplified variable region fragments were cloned into the pCR2.1-TOPOvector (Invitrogen) and DNA sequences determined. Based on thedetermined variable region sequences for the 1-1F11 antibody, thevariable heavy sequence was PCR amplified using the pCR2.1-TOPO clonedvariable region as template and an equimolar mix of 5′ and 3′ primers(SEQ ID NO:42 and SEQ ID NO:43). The variable light sequence was PCRamplified using a similar strategy. The pCR2.1-TOPO cloned variableregion was used as template and an equimolar mix of 5′ and 3′ primers(SEQ ID NO:44 and SEQ ID NO:45). The variable regions were joined by a(G₄S)₃ linker; ScFv genes were assembled and PCR amplified using anequimolar mix of the above specific variable heavy and variable lightPCR products and an equimolar mix of 5′ heavy primer (SEQ ID NO:42) andthe 3′ light primer (SEQ ID NO:45). PCR product was cloned intoInvitrogen pCR2.1-TOPO (Invitrogen) to yield pRG1192. The sequence wasconfirmed before sub-cloning the 747 bp AscI/SrfI to fuse the ScFv geneto the N-terminus of a DNA encoding the human IgG1 Fc fragment (hFc), orthe 756 bp AscI/NotI restriction fragments to fuse the same ScFv to theC-terminus of a DNA encoding hFc.

The 2-1G3 hybridoma was also found to express an antibody capable ofinducing phosphorylation of the Tie-1 receptor in RAECs. Messenger RNAwas isolated and variable heavy cDNA synthesized as described above withequimolar amounts of primers from the from the Wright primer set(Morrison et al. (1995) supra) that included the 5′ heavy chain primers(SEQ ID NO:35-37) and the 3′ primer (SEQ ID NO:33). Similarly, the lightchain variable regions were amplified from cDNA with equimolar amountsof the 5′ heavy chain primers (SEQ ID NO:38-41) and the 3′ primer (SEQID NO:34). The amplified variable region fragments were cloned into thepCR2.1-TOPO vector (Invitrogen) and the DNA sequences were determined.

Based on the determined variable region sequences for the 2-1G3antibody, the variable heavy sequence was PCR amplified using thepCR2.1-TOPO cloned variable region as template and an equimolar mix of5′ and 3′ primers (SEQ ID NO:46 and SEQ ID NO:47). The variable lightsequence was PCR amplified using a similar strategy. The pCR2.1-TOPOcloned variable region was used as template and an equimolar mix of 5′and 3′ primers (SEQ ID NO:48 and SEQ ID NO:49). The variable regionswere joined by a (G4S)3 linker; ScFv genes were assembled and PCRamplified using an equimolar mix of the above specific variable heavyand variable light PCR products and an equimolar mix of 5′ 2-1G3 heavyprimer (SEQ ID NO:46) and the 3′ light primer (SEQ ID NO:49). PCRproduct was cloned into Invitrogen pCR2.1-TOPO (Invitrogen) to yieldpRG1198. The sequence was confirmed before sub-cloning the 738 bpAscI/SrfI to fuse the ScFv gene to the N-terminus of a DNA encoding thehFc fragment or the 747 bp AscI/NotI restriction fragments to fuse thesame ScFv to the C-terminus of a DNA encoding hFc.

Example 8 Construction of Monospecific and Bispecific Activating Dimers

Two types of ScFv-based chimeric molecules were constructed to assessthe ability of ScFv-based molecules to activate the rTie-1 receptor. Onetype of molecule used a single ScFv fused to both the N-terminus and theC-terminus of hFc, the consequence of which was a monospecifictetravalent molecule capable of binding rTie-1. This molecule should becapable of simultaneously binding four rTie-1 molecules. The plasmidpTE778 encodes the gene for ScFv_(1-1F11)-Fc-ScFv_(1-1F11) (SEQ IDNO:50) and contains the mROR1 signal peptide and CMV-MIE promoter. Theprotein was expressed and purified as described above.

Construction of an ScFv-Fc-ScFv molecule where the two ScFv domains arederived from two different non-competing anti-rTie-1 antibodies isexpected to yield a molecule capable of clustering more than fourreceptors, in contrast to the ScFv_(1-1F11)-Fc-ScFv_(1-1F11) describedabove, which can cluster only four receptors. It was determined byBIAcore analysis that the binding of the 1-1F11 antibody did not blockbinding of 2-1G3 to rTie-1, and 1-1F11 binding first did not blockbinding of 2-1G3. Consequently, ScFv molecules made from theseantibodies should be capable of clustering more than four receptors. Toconstruct a bispecific tetravalent ScFv-based molecule, the ScFv_(2-1G3)gene was used in combination with the ScFv_(1-1F11) gene to yieldScFv_(2-1G3)-Fc-ScFv_(1-1F11) (SEQ ID NO:51). Both constructs wereexpressed and purified as described above.

The following Tie-binding proteins were also made: a Tie-binding proteinthat is a dimer of a polypeptide that has an ScFv that binds rat Tie-1linked to a multimerizing component that is an Fc portion, linked to anFD1 (ScFv_(1-1F11)-Fc-FD1) (SEQ ID NO:52); and a Tie-binding proteinthat is a dimer of a polypeptide that has an ScFv that binds rat Tie-1linked to a multimerizing component that is an Fc portion, linked to anFD2 (ScFv_(1-1F11)-Fc-FD2) (SEQ ID NO:53).

Example 9 Activity of Activating Dimers

ScFv_(1-1F11)-Fc-FD1 was tested for its ability to activate rTie-1 andrTie-2 in a RAEC assay as described herein. Exposure ofScFv_(1-1F11)-Fc-FD1 to RAECs bearing rTie-1 and rTie-2 resulted in ahigh degree of phosphorylation of both Tie-1 and Tie-2, as measured byphosphotyrosine blot of immunoprecipitates generated using anti-Tie-1 oranti-Tie-2. Phosphotyrosine blots showed a level of phosphorylation ofboth Tie-1 and Tie-2 that appeared to be at least double thephosphorylation exhibited by exposing the cells to ScFv_(1-1F11)-Fc-FD2and at least 10-fold or more over a mock challenge (i.e., no antibody);in the same assay, a monospecific antibody that binds rTie-1 showedlittle to no phosphorylation of either Tie-1 or Tie-2.

ScFv_(1-1F11)-Fc-FD1 was also compared in a RAEC assay with thefollowing Tie-binding proteins: ScFv_(B2)-Fc-FD1, ScFv_(B2)-Fc-FD2,ScFv_(1-1F11)-Fc-ScFV_(B2), ScFv_(1-1F11)-Fc-FD2, and FD1-Fc-FD1. Blotsof the Tie-1 phosphorylation assays revealed that ScFv_(1-1F11)-Fc-FD1resulted in the highest phosphorylation of Tie-1 for Tie-bindingproteins tested and among the highest activation of Tie-2 for theTie-binding proteins tested.

What is claimed is:
 1. An in vitro method of activating a Tie-1 receptorcomprising: exposing a cell that bears a Tie-1 receptor to a dimerformed from two fusion polypeptides of the form (A)-M-(A′), whereincomponents A and A′ are each a single chain variable fragment (scFv)antibody capable of binding Tie-1, and component M is a multimerizingcomponent comprising an Fc domain of IgG, wherein said dimer binds toand clusters four or more Tie-1 receptors.
 2. The method of claim 1,wherein A and A′ are different scFvs.
 3. The method of claim 1, whereinM comprises an Fc domain of human IgG1.
 4. The method of claim 1,wherein each of the two fusion polypeptides comprisesscFv_(2-1G3)-Fc-scFv_(1-1F11) (SEQ ID NO: 51).
 5. The method of claim 1,wherein each of the two fusion polypeptides comprisesscFv_(1-F11)-Fc-scFv_(1-1F11) (SEQ ID NO: 50).
 6. An in vitro method ofactivating a Tie-1 receptor comprising: exposing a cell that bears aTie-1 receptor to a dimer formed from two fusion polypeptides of theform (A)-M-(A′), wherein component A is a single chain variable fragment(scFv) antibody capable of binding Tie-1, component M is a multimerizingcomponent comprising an Fc domain of IgG, and component A′ is afibrinogen domain of Ang1 or Ang2, wherein said dimer binds to andclusters four or more Tie-1 receptors.
 7. The method of claim 6, whereincomponent A′ is a fibrinogen domain of Ang1.
 8. The method of claim 6,wherein component A′ is a fibrinogen domain of Ang2.
 9. The method ofclaim 6, wherein M comprises an Fc domain of human IgG1.
 10. The methodof claim 6, wherein each of the two fusion polypeptides comprisesscFv_(1-1F11)-Fc-FD1 (SEQ ID NO: 52).
 11. The method of claim 6, whereineach of the two fusion polypeptides comprises scFv_(1-1F11)-Fc-FD2 (SEQID NO: 53).
 12. An in vitro method of activating a Tie-2 receptorcomprising: exposing a cell that bears a Tie-2 receptor to a dimerformed from two fusion polypeptides of the form (A)-M-(A′), whereincomponents A and A′ are each a single chain variable fragment (scFv)antibody capable of binding Tie-2, and component M is a multimerizingcomponent comprising an Fc domain of IgG, wherein said dimer binds toand clusters four or more Tie-2 receptors.
 13. The method of claim 12,wherein A and A′ are different scFvs.
 14. The method of claim 12,wherein M comprises an Fc domain of human IgG1.
 15. The method of claim12, wherein the dimer is formed from two fusion polypeptides eachcomprising scFv_(A12A)-Fc-scFv_(B2) (SEQ ID NO: 28).
 16. The method ofclaim 12, wherein the dimer is formed from two fusion polypeptides eachcomprising scFv_(B2)-Fc-scFv_(B2) (SEQ ID NO: 29).
 17. An in vitromethod of activating a Tie-2 receptor comprising: exposing a cell thatbears a Tie-2 receptor to a dimer formed from two fusion polypeptides ofthe form (A)-M-(A′), wherein component A is a single chain variablefragment (scFv) antibody capable of binding Tie-2, component M is amultimerizing component comprising an Fc domain of IgG, and component A′is a fibrinogen domain of Ang1 or Ang2, wherein said dimer binds to andclusters four or more Tie-2 receptors.
 18. The method of claim 17,wherein component A′ is a fibrinogen domain of Ang1.
 19. The method ofclaim 17, wherein component A′ is a fibrinogen domain of Ang2.
 20. Themethod of claim 17, wherein M comprises an Fc domain of human IgG1. 21.The method of claim 17, wherein each of the two fusion polypeptidescomprises scFv_(B2)-Fc-FD1 (SEQ ID NO: 30).
 22. The method of claim 17,wherein each of the two fusion polypeptides comprises scFv_(B2)-Fc-FD2(SEQ ID NO: 31).